I have tried Trizol and Quiagen RNEasy kits to isolate RNA from waterfowl feces (I added influenza virus in serial dilutions to some feces samples as a positive control). However none of the samples was positive on RT-PCR (controls with RNA isolation from same amount of pure virus culture without feces were clearly positive). Does someone have an experience of detection and isolation of influenza virus from bird feces?
Could it be that your starting quantity of faeces at the start of the extraction is too high? You could try to take a faecal swab and dip that into 1ml of PBS and mix, then continue taking 200µl of supernatans and continue the extraction protocol.
You may need to use a stool-specific kit because there are many inhibitors in stool samples. For example, Qiagen's RNeasy PowerMicrobiome Kit
Print. I recommend contacting Qiagen directly - their tech support is good for finding the right kit for you.
If you still have the extracts from the Trizol/RNeasy kits, I recommend making a dilution series on the extract and adding them to PCR (where each PCR reaction is spiked with influenza DNA and should turn positive if no inhibitors are present).
Try serial diluting your sample and then run RT-qPCR for each dilution and see which dilution gives you a positive result. In my opinion the problem you are facing is PCR inhibition due to inhibitors present in your sample or you are using to much starting material for your RNA extraction. Try using 200 mg of your stool sample directly into your lysis buffer vortex and incubate as mentioned in your extraction manual but for longer duration until the sample completely dissolves in your lysis buffer. You can vortex in between the incubation step which will aid in dissolving.
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